Over the past two decades, reactive oxygen species have been implicated as critical mediators of oxidative processes and disease mechanisms under physiological conditions. Therefore, it is of critical importance to have a direct technique capable of identifying free radicals at their site of formation in systems ranging from chemical to enzymatic reactions, and cellular to in vivo systems so that the mechanisms and processes underlying the oxidative damage in biological systems can be understood. The overall objective of this proposal is to develop novel spin traps with improved properties that can be applied to study oxidative stress in biological systems using electron paramagnetic resonance (EPR) spectroscopy. The specific monitoring of the formation of biologically relevant reactive oxygen species such as ?OH, O2?-, ROO? or RS? is achieved by the distinctive EPR spectral profile they give after addition to spin traps to form a persistent radical adduct. The most commonly used spin traps, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO), are limited by their poor efficiency of trapping superoxide radical anion, short half-life of the radical adduct formed in biological milieu, cytotoxicity, sensitivity and target specificity. Our aim is to overcome such limitations by employing an interdisciplinary approach in spin trap development that encompasses theoretical prediction, organic synthesis, kinetic determination, toxicity, and ultimately, their experimental application to identify and detect free radical formation in in vitro and in vivo systems at their site of formation. The synergistic application of these technologies will provide an optimal strategy to provide new materials which can be effective in probing the role of reactive oxygen species in biological mechanisms.